A Single L- to D-Substitution in Piscidin-2s Creates an N-Terminal Aromatic Anchor That Enhances Membrane Interaction and Antibacterial Activity

Stereochemical modification represents a powerful strategy for optimizing the therapeutic potential of antimicrobial peptides. Herein, we present a comprehensive structural and biophysical characterization of ecPis-2s and its epimerized analog, d-ecPis-2s, featuring a single d-F substitution. Although both peptides possess identical net charges and global hydrophobicity, our results reveal that the d-substitution acts as a conformational switch altering membrane interaction dynamics and antimicrobial efficacy. CD and solution NMR spectroscopy demonstrate that d-ecPis-2s maintains a similar overall helical character but adopts a distinctive N-terminal structural bend. Structural refinements and topology studies based on solid-state NMR elucidated that d-configuration at position 2 promotes a deeper sequestration of the N-terminal aromatic triad (F1−F3) into the hydrophobic membrane core. This aromatic anchor drives the peptide backbone and consecutively the aromatic side chains into the lipid bilayer, enhancing membrane tension, pore-forming kinetics and lipid packing disruption, which correlates directly with increased antimicrobial activity.